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118 Cell-Penetrating Peptides: Processes and Applications
following a 30-min incubation; on average, 20 times more 125 I-SKP was associated
with cells compared to a control peptide that lacked the MTS. The addition of the
proteases Pronase and trypsin to the medium after incubation did not decrease the
amount of radioactivity associated with the cells, indicating that the SKP peptide
was at the very least buried in the membrane, if not present in the cytoplasm, and
therefore inaccessible to the enzymes. Furthermore, cellular uptake was not inhibited
by unlabeled SKP, thus excluding the possibility that the uptake was receptor mediated.
It was also concluded that the peptide translocation was not reliant on large
stores of ATP after it was shown that incubation with ATP-depleted cells did not
prevent peptide uptake.
Once the translocation activity of the kFGF h-region had been confirmed, a
peptide containing both the kFGF h-region and the nuclear localization sequence
(NLS) of the nuclear factor-kappa B (NF-κB) p50 subunit was synthesized. The
purpose was to produce a peptide that could translocate into intact cells and aid in
the study of the role of the NF-κB NLS in the intracellular trafficking of this
transcription factor. 33 The import of this chimeric peptide, termed SN50, into murine
endothelial LE-II cells was monitored by confocal microscopy using an indirect
immunofluorescence detection protocol. The import of SN50 was determined to be
concentration-, time-, and temperature dependent, with maximum fluorescence
observed after incubation at 37°C for 30 to 60 min at a concentration of 50 to
100 µg/ml.
Of equal importance was the biological activity of SN50; the peptide successfully
competed with NF-κB complexes for the cellular machinery responsible for agonistinduced
nuclear translocation of NF-κB. The inhibition of NF-κB nuclear translocation
was concentration dependent, with the maximum inhibition occurring when
the extracellular concentration of SN50 reached 50 µg/ml (18 µ M). This is the first
example of the ability of an MTS to transport a functional domain into intact cells.
The SN50 peptide was used in a related study to delineate the mechanisms by
which the nuclear import machinery recognizes the transcription factors NF-κB,
AP-1, NFAT, and STAT1, each of which differs in NLS sequence. 37 Each of these
transcription factors plays a critical role in the expression of genes important for
cell growth, differentiation, and adhesion. 38,39 It was found that administration of
SN50 to the medium containing human Jurkat T lymphocytes resulted in the uptake
of the peptide by the activated cells. The imported SN50 was shown to inhibit nuclear
import of NF-κB, AP-1, and NFAT at a high dose of 210 µg/ml in Jurkat cells. In
other studies, however, lower doses of SN50 selectively and specifically inhibited
NF-κB translocation in primary cells. For example, in primary human peripheral
blood lymphocytes, lower doses of SN50 peptide (37.5 µg/ml) were shown to inhibit
NF-κB only, without inhibiting AP-1 and NFAT. 40
The ability of SN50 to inhibit the import and nuclear function of NF-κB without
the use of disruptive methods led to the development of a new strategy to exert an
immunosuppressive effect on activated T lymphocytes. Similar methods have been
used to study factors critical to the growth and differentiation of many cell types as
highlighted by the following examples.